Conformational Ensemble of Glutamate Transporters: Structure and Ionic Modulatio

描述（由申请人提供）：兴奋性氨基酸转运蛋白（EAATs）在神经传递后清除突触间隙中的谷氨酸，并负责所有细胞类型对酸性和中性氨基酸的摄取。在已有的电化学梯度的驱动下，EAATs将底物摄取与钠离子和质子的共输运以及钾的反输运结合起来。先前对一种细菌同源物——阔氏焦球菌GltPh的晶体学研究表明，这种转运体在细胞外溶液中处于外向状态，与底物结合位点可接触，当与底物结合时被封闭，当与阻滞剂结合时被暴露。这些结构解释了底物和钠离子如何从细胞外溶液到达它们的结合位点，但没有回答这些溶质如何跨膜转运并释放到细胞质中。人们认为，转运体的构象转变为向内的状态，在这种状态下，底物结合位点可以被细胞内溶液所接近，这是底物解离进入细胞质的先决条件。在目前的应用中，我们建议使用双半胱氨酸诱变和交联来描述转运体进入内向状态时发生的结构重排，并探索该过程的动力学。我们进一步建议通过半胱氨酸交联来确定在内向状态和可能在其他中间运输状态下共价约束的转运蛋白分子的晶体结构。最后，我们将采用位点定向诱变与生化和结构分析相结合的方法来探测GltPh和eaat中离子结合和渗透位点的位置

DESCRIPTION (provided by applicant): The excitatory amino acid transporters (EAATs) clear glutamate from the synaptic cleft following rounds of neurotransmission and are responsible for the uptake of acidic and neutral amino acids by all cell types. Driven by pre-existing electrochemical gradients, EAATs couple substrate uptake to the co-transport of sodium ions and protons and to the counter-transport of potassium. Previous crystallographic studies on a bacterial homologue from Pyrococcus horikoshii, GltPh have revealed the transporter in the outward facing states with the substrate-binding site accessible to the extracellular solution and either occluded when bound to a substrate or exposed when bound to a blocker. These structures have explained how the substrate and sodium ions reach their binding sites from the extracellular solution but left unanswered how these solutes are translocated across the membrane and released into the cytoplasm. It is believed that a conformational transition of the transporter into an inward facing state, in which the substrate-binding site is accessible to the intracellular solution, is a prerequisite for the substrate dissociation into the cytoplasm. In the current application, we propose to use double cysteine mutagenesis and cross-linking to delineate the structural re- arrangements that occur upon transition of the transporter into the inward facing state and to probe the dynamics of the process. We further propose to determine the crystal structures of the transporter molecules covalently constrained in the inward facing state and possibly in other intermediate transport states by cysteine cross-linking. Finally, we will employ the site directed mutagenesis in conjuncture with biochemical and structural analyses to probe the location of the ion binding and permeation sites in GltPh and, by analogy, in EAATs